Direct-write lithographic patterning or printing of biological structures is important for many reasons. For example, direct-write lithographic patterning provides fabrication of microscopic and nanoscopic patterns with extraordinary complexity and offers routes to important devices in the life sciences such as gene chips, proteomic arrays, and other diagnostic tools. Important biological nanostructures in life science applications include oligonucleotides, DNA, peptides, proteins, and viruses. In particular, microarrays of biomolecules such as DNA and proteins have proven useful as high throughput screening tools in proteomics, genomics, and the identification of new pharmaceutical compounds. For example, DNA microarrays can be used to probe gene expression and in panel assays for research- and clinical-based diagnostics. Arrays of proteins have been used to ask and answer important questions regarding the interactions of cells with underlying substrates. As the complexity of these arrays and corresponding number of features increase, the ability to reduce feature size becomes more important, especially since the area occupied by an array will affect the amount and volume of a sample that can he used with a particular chip. Therefore, arrays with smaller and more densely packed features are becoming increasingly attractive. In addition, if one can fabricate such structures with features that have nano- rather than macroscopic dimensions, one can enable new screening technologies and begin to address important fundamental questions regarding biomolecular recognition that are not addressable with microarrays. Indeed, bio-recognition is generally a nanoscopic rather than microscopic phenomenon.
In particular, direct-write nanolithographic patterning and printing of peptide- and protein-based nanostructures is important in a variety of areas including, for example, proteomics, diagnostics, and materials science. Such methods would allow one to fabricate patterns of peptide and protein nanostructures with extraordinary complexity, offering routes to important tools in the life sciences such as protein libraries, protein chips, and proteomic arrays, and templates that can be used by chemists and material scientists to build ordered two and three-dimensional functional architectures.
Direct-write nanolithographic patterning or printing offers advantages over competing approaches such as indirect patterning methods. In a typical indirect method, an intermediate compound is first patterned which serves as a template for the biological nanostructure design. Unlike photolithographic methods, the direct-write method has no need for a resist and easier processing and fabrication procedures. As a result, known direct-write nanolithographic printing methods have become powerful tools in nanotechnology. Some of these methods use one or more nanoscopic tips, such as scanning probe microscope (SPM) tips including atomic force microscope (AFM) tips. In some embodiments, the tips are used to deliver one or more patterning compounds onto a substrate surface from the tips. The result is the ability to generate detailed, stable patterns at high resolution and nanoscale dimensions over a wide variety of shapes in serial or parallel modes. Potential applications range beyond biotechnology and pharmaceutical industries, extending to semiconductor and computer technologies as well.
Despite its importance, direct-write patterning or printing methods can pose challenges not encountered in the indirect methods. For example, challenges can arise in transporting high-molecular-weight biomolecules from a coated tip to a substrate and the need for bio-compatible patterning conditions. High resolution and patterning speed are important which should not be sacrificed in patterning biomolecules.
Although some promising advances have been made in making protein patterns with features with nanoscopic dimensions, challenges remain, particularly with patterns below about 200 nm in lateral dimension. For example, protein nanopatterns generally have been made by indirect methods that either involve resists or prefabricated chemical affinity templates. These templates direct the assembly of a single protein structure from solution onto a set of nanoscopic features on a surface of interest. Often, the bio-recognition properties and control over feature size on the sub-200 nm scale are not demonstrated. In addition, to be able to generate nanoarrays of multicomponent systems, a requisite for many of the anticipated applications of nanoarrays, it is important that new surface analytical tools as well as the complementary chemistry be developed for directly placing a set of different protein structures on a surface of interest with nanoscale resolution, high registration alignment capabilities, and control over the biological activity of the resulting structures.
In addressing these challenges, one promising direct-write patterning approach is being developed by the Mirkin group at Northwestern University and NanoInk, Inc. under the name DIP-PEN NANOLITHOGRAPHY™ (DPN)™ patterning and printing (proprietary trademarks of NanoInk, Inc., Chicago, Ill.). In a typical DPN™ printing application, a patterning compound is transported from a nanoscopic tip, preferably an AFM tip, to a substrate to form a desired, stable, useful nanostructure. Basic and novel features of the DPN™ process include the absence of resists, masks, and destructive patterning. Improved patterning methods are needed, however, to maximize commercial applications.